猪流行性腹泻病毒YZ2016全基因组测序与稳定性分析
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摘要
2016年我国江苏扬州某规模化养猪场暴发严重腹泻,本实验室通过RT-PCR确定其病原为猪流行性腹泻病毒(PEDV)。将阳性样品接种Vero细胞,盲传5代后出现典型病变,并通过RT-PCR及间接免疫荧光试验确定成功分离到PEDV毒株,命名为PEDVYZ2016。采用分段重叠法对分离株基因组扩增测序,获得其全长序列。遗传进化分析显示PEDV YZ2016与近年流行毒株亲缘关系较近,与经典毒株亲缘关系较远。PEDV YZ2016与BJ-2011-1、LZW全基因组核苷酸序列一致性可达99. 2%,与BJ-2011-1、OKN-1/JPN/2013 S基因推导氨基酸序列一致性可达98.5%。此外,分离株S基因发生多处点突变,导致其抗原表位及糖基化位点均发生部分改变。同时,序列比对分析显示不同代次的S、ORF3、N基因核苷酸突变率均极低。结果表明,本研究分离的PEDV YZ2016株属流行变异毒株,且在传代过程中遗传稳定性良好。
In 2016,severe diarrhea occurred on a large-scale pig farm in Yangzhou,Jiangsu Province,China.The pathogen was determined to be porcine epidemic diarrhea virus(PEDV) by RT-PCR.The Vero cells infected with the positive sample were passaged five times and then subjected to RT-PCR and indirect immunofluorescence assays(IFA).The results demonstrated that a new PEDV strain was successfully isolated and named PEDV YZ2016.Lately,the isolate was cloned and sequenced for the whole genome sequence.Phylogenetic analysis showed that PEDV YZ2016 was closely related to the epidemic strains in recent years,but far from the classical strains.The identity of whole-genome nucleotide sequences of PEDV YZ2016 with BJ-2011-1 and LZW was up to 99.2%,and the identity of S gene derived-amino acid sequences of PEDV YZ2016 with BJ-2011-1 and OKN-1/JPN/2013 was up to 98.5%.In addition,multiple point mutations of S gene resulted in important changes in antigen epitopes and glycosylation sites.Meanwhile,alignment analysis showed that the nucleotide mutation rates of S,ORF3 and N genes of different generations were extremely low.In conclusion,PEDV YZ2016 was an epidemic strain,and genetically stable during the passaging.
In 2016,severe diarrhea occurred on a large-scale pig farm in Yangzhou,Jiangsu Province,China.The pathogen was determined to be porcine epidemic diarrhea virus(PEDV) by RT-PCR.The Vero cells infected with the positive sample were passaged five times and then subjected to RT-PCR and indirect immunofluorescence assays(IFA).The results demonstrated that a new PEDV strain was successfully isolated and named PEDV YZ2016.Lately,the isolate was cloned and sequenced for the whole genome sequence.Phylogenetic analysis showed that PEDV YZ2016 was closely related to the epidemic strains in recent years,but far from the classical strains.The identity of whole-genome nucleotide sequences of PEDV YZ2016 with BJ-2011-1 and LZW was up to 99.2%,and the identity of S gene derived-amino acid sequences of PEDV YZ2016 with BJ-2011-1 and OKN-1/JPN/2013 was up to 98.5%.In addition,multiple point mutations of S gene resulted in important changes in antigen epitopes and glycosylation sites.Meanwhile,alignment analysis showed that the nucleotide mutation rates of S,ORF3 and N genes of different generations were extremely low.In conclusion,PEDV YZ2016 was an epidemic strain,and genetically stable during the passaging.
引文
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[15]HOFMANN M,WYLER R.Quantitation,biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus(PEDV)[J].Veterinary Microbiology,1989,20(2):131-142.
[16]SHI W,JIA S,ZHAO H Y,et al.Novel approach for isolation and identification of porcine epidemic diarrhea virus(PEDV)strain NJ using porcine intestinal epithelial cells[J].Viruses,2017,9(1):19.
[17]韩莎莎,张玉娟,时庆贺,等.河南省2014-2015年猪流行性腹泻病毒流行猪遗传进化分析[J].中国兽医学报,2017,37(6):976-983.HAN Sha-sha,ZHANG Yu-juan,SHI Qing-he,et al.Genetic variation of porcine epidemic diarrhea virus prevailing in Henan Province during 2014-2015[J].Chinese Journal of Veterinary Science,2017,37(6):976-983.(in Chinese)
[2]PENSAERT M,DEBOUCK P.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243-247.
[3]DEBOUCK P,PENSAERT M.Experimental infection of pigs with a new porcine enteric coronavirus,CV777[J].American Journal of Veterinary Research,1980,41(2):219-223.
[4]CHEN X,YANG J X,YU F S,et al.Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus(PEDV)samples from field cases in Fujian,China[J].Virus Genes,2012,45(3):499-507.
[5]SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerging Infectious Diseases,2012,18(1):161-163.
[6]GE F F,YANG D Q,JU H B,et al.Epidemiological survey of porcine epidemic diarrhea virus in swine farms in Shanghai,China[J].Archives of Virology,2013,158(11):2227-2231.
[7]HOANG H,KILLIAN M L,MADSON D M,et al.Full-length genome sequence of a Plaque-cloned virulent porcine epidemic diarrhea virus isolate(USA/Iowa/18984/2013)from a midwestern U.S.swine herd[J].Genome Announcements,2013,1(6):e01049-13.
[8]MARTHALER D,JIANG Y,OTTERSON T,et al.Complete genome sequence of porcine epidemic diarrhea virus strain USA/Colorado/2013 from the United States[J].Genome Announcements,2013,1(4):e00555-13.
[9]CHEN J F,WANG C B,SHI H Y,et al.Molecular epidemiology of porcine epidemic diarrhea virus in China[J].Archives of Virology,2010,155(9):1471-1476.
[10]BOSCH B J,VANDERZEE R,DEHAAN C A,et al.The coronavirus spike protein is a classⅠvirus fusion protein:structural and functional characterization of the fusion core complex[J].Journal of Virology,2003,77(16):8801-8811.
[11]SUN D B,FENG L,SHI H Y,et al.Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein[J].Veterinary Microbiology,2008,131(1-2):73-81.
[12]CHANG S H,BAE J L,KANG T J,et al.Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus[J].Molecules and Cells,2002,14(2):295-299.
[13]CRUZ D J,KIM C J,SHIN H J.The GPRLQPY motif located at the carboxy-terminal of the spike protein induces antibodies that neutralize porcine epidemic diarrhea virus[J].Virus Research,2008,132(1-2):192-196.
[14]HUANG Y W,DICKERMAN A W,PINEYRO P,et al.Origin,evolution,and genotyping of emergent porcine epidemic diarrhea virus strains in the United States[J].American Society for Microbiology,2013,4(5):e00737-13.
[15]HOFMANN M,WYLER R.Quantitation,biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus(PEDV)[J].Veterinary Microbiology,1989,20(2):131-142.
[16]SHI W,JIA S,ZHAO H Y,et al.Novel approach for isolation and identification of porcine epidemic diarrhea virus(PEDV)strain NJ using porcine intestinal epithelial cells[J].Viruses,2017,9(1):19.
[17]韩莎莎,张玉娟,时庆贺,等.河南省2014-2015年猪流行性腹泻病毒流行猪遗传进化分析[J].中国兽医学报,2017,37(6):976-983.HAN Sha-sha,ZHANG Yu-juan,SHI Qing-he,et al.Genetic variation of porcine epidemic diarrhea virus prevailing in Henan Province during 2014-2015[J].Chinese Journal of Veterinary Science,2017,37(6):976-983.(in Chinese)